The present invention relates to monoclonal antibodies, secreted by hybridoma cell lines, that are directed against an epitope of the extracellular domain of human VEGF-receptor KDR (kinase insert domain containing receptor), to methods of determining human VEGF-receptor KDR in cell lysates or tissue analysates and to the use of the antibodies in analytical assays, in diagnostics and as carrier molecules for therapeutic substances.
New vessels form as capillaries which sprout from existing small vessels. This process, known as angiogenesis, takes place in response to certain signals (Alberts et al., Molekularbiologie der Zelle, Verlag Chemie, Weinheim).
Angiogenesis is a process that is meticulously controlled by the interaction of vascular endothelial growth factor (vascular endothelial growth factor) and its corresponding highly affine KDR-receptor on endothelial cells. KDR has been characterised as a transmembranal tyrosine kinase receptor of sub-type 5 which serves as a key regulator of vascular endothelial cell development during embryogenesis and cell regeneration (Cancer and Metastasis Reviews 15: 159-163, 1996). Furthermore, the dysfunction of that normally meticulously regulated ligand-receptor interaction results in impairment of the angiogenesis process, which is a feature of many illnesses. Particularly important is that the growth of tumours and their metastases has proved to be angiogenesis-dependent to a high degree.
A monoclonal antibody having a limited reaction pattern is already known. That antibody is directed against the extracellular domain of mouse KDR-homologous flk-1 and is capable of neutralising the VEGF stimulation of a chimeric flk-1/fms-receptor that is expressed in transfected 3T3 cells (Rockwell et al., Molecular and Cellular Differentiation, 3 (1): 91-109 (1995)).
Monoclonal antibodies that can be used for a broad spectrum of analytical assays and methods of determining human VEGF-receptor KDR in cell lysates and tissue analysates using such antibodies have not been known hitherto.
The aim of the present invention is to provide monoclonal antibodies secreted by hybridoma cell lines. The monoclonal antibodies according to the invention can be used for a broad spectrum of analytical procedures and assays, in diagnostics and as carrier molecules for therapeutic substances.
There have now been prepared monoclonal antibodies that are directed against an epitope located within extracellular domains 1 to 7 of human VEGF-receptor KDR.
The present invention relates especially to monoclonal antibodies that are directed against an epitope located within extracellular domains 6 and 7 of human VEGF-receptor KDR.
The monoclonal antibodies AM 2-7-9, AM 2-10-1, AM 5-1-2 AM 5-10-13 and AM 2-4-1 are preferred.
The monoclonal antibodies AM 2-7-9 and AM 2-10-1 are especially preferred.
Those antibodies are directed specifically against an epitope that is located within extracellular domains 6 and 7 of human VEGF-receptor KDR.
The antibodies according to the invention exhibit a high degree of selectivity in a broad spectrum of analytical procedures and assays.
The monoclonal antibodies according to the invention have the advantage that they neither interfere with the ligand-binding domain nor activate KDR after binding.
The antibodies according to the invention are also valuable for diagnostic purposes.
For example, the monoclonal antibodies according to the invention can be used in Western blots, immunoprecipitation, ELISA, FACS analysis and in indirect immunofluoresence microscopy.
A further use of the monoclonal antibodies is in immunohistochemistry.
The monoclonal antibodies can also be used in screening for small agonistic and antagonistic molecules and in the detection of mutant receptor subtypes.
The monoclonal antibodies according to the invention can also be used in diagnostics, it being possible to couple them in combination with a suitable contrast-enhancing substance. The antibodies react with an epitope that is not located within the ligand-binding side of the KDR and is therefore capable of reacting with all KDR-receptor populations.
A preferred contrast-enhancing substance that can be coupled to the antibodies is technetium-99.
The monoclonal antibodies according to the invention can also be used in angiogenesis-dependent phenotypes, such as tumours and metastases thereof, rheumatoid arthritis or psoriasis, or pathological symptoms resulting therefrom, when coupled with suitable toxic substances.
Suitable toxic substances are adequately known to the person skilled in the art and are described, for example, in Byers and Baldwin, Immunology (1988), 65, 329-335 and in Blakey et al., Waldmann H. (ed): Moncclonal Antibody Therapy, Prog. Allergy. Basle, Karger, (1988), vol. 45, 50-90.
The invention relates also to the use of mRNA that codes for the heavy and the light chain of the antibodies according to the invention in the preparation of recombinant xe2x80x9csingle-chain antibodiesxe2x80x9d.
The invention relates also to the use of the recombinant antibodies encoded by mRNA for Western blots, immunoprecipitation, ELISA and FACS analysis, in indirect immunofluoresence microscopy and immunohistochemistry and in screening for small agonistic and antagonistic molecules and in the detection of mutant receptor subtypes.
The invention relates also to a method of determining human VEGF-receptor KDR in cell lysates or tissue analysates, characterised in that
1. the monoclonal antibodies AM 2-7-9, 2-10-1, AM 5-1-2, AM 5-10-13 or AM 2-4-1, as captor antibodies, are coupled in purified form at a concentration of 1-10 xcexcg/ml in coupling buffer on ELISA plates and then excess binding sites are blocked with blocking buffer,
2. cell or tissue analysates are prepared in a suitable lysis buffer,
3. the plates are washed in washing buffer before application of the lysates,
4. the KDR-protein to be determined in the sample is quantified using a recombinant KDR-protein as standard curve,
5. the tissue and cell lysates to be analysed are then introduced into the test system,
6. incubation is carried out at room temperature for 2 hours,
7. the ELISA plates are then washed.
8. determination of the xe2x80x9ccapturedxe2x80x9d KDR is carried out by means of a polyclonal anti-KDR antiserum and finally
9. detection by means of a chromogenic, chemiluminescent or radioactive substance is carried out.
The method can also take place in the form of a kinase test procedure that determines tyrosine-phosphorylated KDR.
In order to allow quantification of the test method, recombinant phosphorylated KDR can be used as the standard protein.
The determination of human VEGF-receptor KDR in cell lysates or tissue analysates can be carried out both qualitatively and quantitatively using the method.
The polyclonal anti-KDR antiserum used in step 8 of the method can be, for example, polyclonal anti-phosphotyrosine antiserum or a monoclonal anti-phosphotyrosine antibody.
The detection carried out in step 9 of the method can preferably be carried out with peroxidase-labelled secondary antibodies and suitable chromogenic or chemiluminescent substrates.
The antiserum used can be, for example, from goats or rabbits or from sheep, rats or donkeys, either directly in the form of enzyme-labelled immunoglobulin or indirectly by means of enzyme-labelled anti-goat, anti-rabbit, anti-sheep, anti-rat or anti-donkey antibodies. Either alkaline phosphatase or peroxidase is used for that purpose.
The ELISA plates used in step 1 of the method can be antibody-coated and blocked.
The lysis buffer used in step 2 of the method contains 1-5 mM divalent ions and 1-15% glycerol.
The lysis buffer preferably contains 1-2 mM divalent ions and 5-12% glycerol.
A lysis buffer that contains 1.5 mM divalent ions and 10% glycerol is especially preferred.
A preferred divalent ion is, for example, magnesium.
The washing and dilution buffers used in the method can be any buffers known to the person skilled in the art for that purpose.
Preferred washing buffers are those containing PBS with 0.05% detergent and 0.1% bovine serum albumin.
Preferred dilution buffers are those containing 1% bovine serum albumin.